Chemokine panec-2 polynucleotides, polypeptides, antibodies and methods of making and using them

ABSTRACT

The present invention provides nucleotide and amino acid sequences that identify and encode novel expressed chemokines (PANEC-1 and PANEC-2) from human pancreas cells. The present invention also provides for antisense molecules to the nucleotide sequences which encode PANEC-1 and PANEC-2, expression vectors for the production of purified PANEC-1 and PANEC-2, antibodies capable of binding specifically to PANEC-1 and PANEC-2, hybridization probes or oligonucleotides for the detection of PANEC-1- or PANEC-2- encoding nucleotide sequences, genetically engineered host cells for the expression of PANEC-1 and PANEC-2, diagnostic tests for chemokine activation based on PANEC-1- and PANEC-2 -encoding nucleic acid molecules and antibodies capable of binding specifically to the protein.

This application is a continuation application of U.S. application Ser.No. 08/390,740, filed Feb. 17, 1995, entitled NEW CHEMOKINES EXPRESSEDIN PANCREAS, all of which applications and patents are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

The pancreas is an elongated organ which lies behind and below thestomach and consists of both exocrine and endocrine tissues. Indescending order, the exocrine portion is divided into lobes, lobules,and functional secretory units known as acini. All acini eventuallydrain into the main pancreatic duct which joins the bile duct from theliver before it empties into the duodenum. Acinar cells comprise 80% ofthe pancreas and secrete enzymes in either inactive or active form whichassist digestion. Epithelial cells of the ductules secrete large amountsof bicarbonate ions and water which neutralize acidic chyme as it leavesthe stomach and enters the duodenum as well as the enzymes for digestingprotein, carbohydrates, and fats.

The most important and abundant proteolytic enzymes are trypsin,chymotrypsin, and carboxypeptidase. The serine proteases, trypsin andchymotrypsin, split whole and partially-digested proteins intopolypeptides of different sizes; then, carboxypeptidase breaks down thepolypeptides into individual amino acids. Several elastases, which arealso serine proteases, and nucleases, which digest nucleic acids, arealso found in the pancreatic juice.

The principal enzyme for digesting carbohydrates in the gut ispancreatic amylase. It hydrolyzes starches, glycogen, and most othernon-cellulosic carbohydrates to form disaccharides and trisaccharides.The main enzymes for fat digestion are pancreatic lipase, cholesterolesterase, and phospholipase. Pancreatic lipase hydrolyzes neutral fatinto fatty acids and monoglycerides. Cholesterol esterase hydrolyzescholesterol esters, and phospholipase removes fatty acid molecules fromphospholipids.

The four molecules which control acinar secretion are acetylcholine andthe hormones, gastrin, cholecystokinin (CCK), and secretin.Acetylcholine is released from the parasympathetic vagus and othercholinergic nerve endings, gastrin is secreted by cells of the stomach,and CCK and secretin are secreted by the upper small intestine. Thegastrointestinal (GI) hormones are absorbed into the blood andtransported to the pancreas where they stimulate acini to secreteenzymes and ductal cells to secrete the sodium bicarbonate and waterwhich washes the pancreatic enzymes into the duodenum.

The endocrine pancreas consists of islets of Langerhans, whose cells areseparated from the exocrine lobules and are distributed throughout thepancreas. The function of the various types of endocrine cells whichmake up the islets is to secrete the hormones which participate in themetabolism of proteins, carbohydrates, and fats.

The major endocrine cells are α, β, and δ cells; the minor cells are Ccells, EC cells, and PP cells. About 15% of the islet cell populationare a cells which are located along the periphery of islets and secretethe hormone glucagon. β cells comprise about 70% of the islet cellpopulation, are located around the center of the islets, and secrete thehormone insulin. δ cells comprise about 10% of the population, arelocated close to a cells and secrete two different hormones,somatostatin and vasoactive intestinal peptide (VIP). C, EC, and PPcells make up the final 5% of the islet cell population. The function ofC cells is unknown, but EC and PP cells secrete serotonin and pancreaticpolypeptide, respectively.

Inflammation of the pancreas or pancreatitis may be classified as eitheracute or chronic by clinical criteria. With treatment, acutepancreatitis can often be cured and normal function restored. Chronicpancreatitis often results in permanent damage. The precise mechanismswhich trigger acute inflammation are not understood. However, somecauses in the order of their importance are alcohol ingestion, biliarytract disease, post-operative trauma, and hereditary pancreatitis. Onetheory provides that autodigestion, the premature activation ofproteolytic enzymes in the pancreas rather than in the duodenum, causesacute pancreatitis. Any number of other factors including endotoxins,exotoxins, viral infections, ischemia, anoxia, and direct trauma mayactivate the proenzymes. In addition any internal or external blockageof pancreatic ducts can also cause an accumulation of pancreatic juicesin the pancreas resulting cellular damage.

As is the case in inflammation of other tissues, leukocytes includingmonocytes, macrophages, basophils, and eosinophils infiltrate theinflamed area of the pancreas. Their primary role is to clean up thesite of the inflammation; however, macrophages may produce powerfuloxidants and proteases which contribute to tissue destruction.Leukocytes also secrete a range of cytokines which recruit other cellsto the area.

The investigation of the critical, regulatory processes by which whitecells proceed to their appropriate destination and interact with othercells is underway. The current model of leukocyte movement ortrafficking from the blood to injured or inflamed tissues comprises thefollowing steps. The first step is the rolling adhesion of the leukocytealong the endothelial cells of the blood vessel wall. This movement ismediated by transient interactions between selectins and their ligands.A second step involves cell activation which promotes a more stableleukocyte-endothelial cell interaction mediated by the integrins andtheir ligands. This stronger, more stable adhesion precipitates thefinal steps of leukocyte diapedesis and extravasation into the tissues.

The chemokine family of polypeptide cytokines possesses the cellularspecificity required to explain leukocyte trafficking in differentabnormal, inflammatory or diseased situations. First, chemokines mediatethe expression of particular adhesion molecules on endothelial cells;and second, they generate gradients of chemoattractant factors whichactivate specific cell types. In addition, the chemokines stimulate theproliferation of specific cell types and regulate the activation ofcells which bear specific receptors. These activities demonstrate a highdegree of target cell specificity.

The chemokines are small polypeptides, generally about 70-100 aminoacids (aa) in length, 8-11 kD in molecular weight and active over a1-100 ng/ml concentration range. Initially, they were isolated andpurified from inflamed tissues and characterized relative to theirbioactivity. More recently, chemokines have been discovered throughmolecular cloning techniques and characterized by structural as well asfunctional analysis.

The chemokines are related through a four-cysteine motif which is basedprimarily on the spacing of the first two cysteine residues in themature molecule. Currently the chemokines are assigned to one of twofamilies, the C-C chemokines (α) and the C-X-C chemokines (β). Althoughexceptions exist, the C-X-C chemokines generally activate neutrophilsand fibroblasts while the C-C chemokines act on a more diverse group oftarget cells which include monocytes/macrophages, basophils,eosinophils, T lymphocytes and others. The known chemokines of bothfamilies are synthesized by many diverse cell types as reviewed inThomson A. (1994) The Cytokine Handbook, 2d Ed. Academic Press, NY. Thetwo groups of chemokines will be described in turn.

At this time, relatively few C-C chemokines have been described, andthey appear to have less N-terminal processing than the C-X-Cchemokines. A brief description of the known human (and/or murine) C-Cchemokines follows. The macrophage inflammatory proteins alpha and beta(MIP-1α and β) were first purified from stimulated mouse macrophage cellline and elicited an inflammatory response when injected into normaltissues. At least three distinct and non-allelic genes encode humanMIP-1α, and seven distinct genes encode MIP-1β.

MIP-1α and MIP-1β consist of 68-69 amino acids which are about 70%identical in their acidic, mature secreted forms. They are bothexpressed in stimulated T cells, B cells and monocytes in response tomitogens, anti-CD3 and endotoxin, and both polypeptides bind heparin.While both molecules stimulate monocytes, MIP-1α chemoattracts the CD-8subset of T lymphocytes and eosinophils, while MIP-1β chemoattracts theCD-4 subset of T lymphocytes. In mouse, these proteins are known tostimulate myelopoiesis.

I-309 was cloned from a human γ-δ T cell line and shows 42% amino acididentity to T cell activation gene 3 (TCA3) cloned from mouse. There isconsiderable nucleotide homology between the 5′ flanking regions ofthese two proteins, and they share an extra pair of cysteine residuesnot found in other chemokines. Such similarities suggest I-309 and TCA3are species homologs which have diverged over time in both sequence andfunction.

RANTES is another C-C chemokine which is expressed in T cells (but not Bcells), in platelets, in some tumor cell lines, and in stimulatedrheumatoid synovial fibroblasts. In the latter, it is regulated byinterleukins-1 and -4, transforming nerve factor and interferon-γ. ThecDNA cloned from T cells encodes a basic 8 kD protein which lacksN-linked glycosylation and is able to affect lymphocytes, monocytes,basophils and eosinophils. The expression of RANTES mRNA issubstantially reduced following T cell stimulation.

Monocyte chemotactic protein (MCP-1) is a 76 amino acid protein whichappears to be expressed in almost all cells and tissues upon stimulationby a variety of agents. The targets of MCP-1, however, are limited tomonocytes and basophils in which it induces a MCP-1 receptor:Gprotein-linked calcium flux (Charo I, personal communication). Two otherrelated proteins (MCP-2 and MCP-3) were purified from a humanosteosarcoma cell line. MCP-2 and MCP-3 have 62% and 73% aa identity,respectively, with MCP-1 and share its chemoattractant specificity formonocytes.

Current techniques for diagnosis of abnormalities in the inflamed ordiseased tissues mainly rely on observation of clinical symptoms orserological analyses of body tissues or fluids for hormones,polypeptides or various metabolites. Patients often manifest no clinicalsymptoms at early stages of disease or tumor development. Furthermore,serological analyses do not always differentiate between invasivediseases and genetic syndromes which have overlapping or very similarranges. Thus, development of new diagnostic techniques comprising smallmolecules such as the expressed chemokines are important to provide forearly and accurate diagnoses, to give a better understanding ofmolecular pathogenesis, and to use in the development of effectivetherapies.

The pancreas is reviewed in Guyton A. C. (1991) Textbook of MedicalPhysiology, W. B. Saunders Co, Philadelphia; and The Merck Manual ofDiagnosis and Therapy, (1992) Merck Research Laboratories, Rahway, N.J.The chemokine molecules are reviewed in Schall T. J. (1994) ChemotacticCytolines: Targets for Therapeutic Development. International BusinessCommunications, Southborough, Mass., pp 180-270; and in Paul W. E.(1993) Fundamental Immunology, Raven Press, New York City (NYC), pp822-826.

SUMMARY OF THE INVENTION

The subject invention provides nucleotide sequences which uniquelyencode two novel human pancreatic proteins. The new genes, which areknown as pancreatic expressed chemokines, or panec-1 and panec-2 (IncyteClones 223187 and 226152), encode polypeptides designated PANEC-1 andPANEC-2, of the C-C chemokine family, and referred to collectively asPANEC.

The invention also comprises diagnostic tests for physiologic orpathologic compromise of the pancreas which include the steps of testinga sample or an extract thereof with panec-1 or panec-2 DNA, fragments oroligomers thereof. Aspects of the invention include the antisense DNAsof panec-1 and panec-2; cloning or expression vectors containing panec-1or panec-2; host cells or organisms transformed with expression vectorscontaining panec-1 or panec-2; a method for the production and recoveryof purified PANEC-1 or PANEC-2 from host cells; and purified proteins,PANEC-1 and PANEC-2.

DESCRIPTION OF THE FIGURES

FIG. 1 displays the nucleotide sequence for panec- 1 (SEQ ID NO:1) andthe predicted amino acid (aa) sequence of the pancreas expressedchemokine, PANEC-1 (SEQ ID NO:2).

FIG. 2 displays the nucleotide sequence for panec-2 (SEQ ID NO:3) andthe predicted amino acid (aa) sequence of the pancreas expressedchemokine, PANEC-2 (SEQ ID NO:4).

FIG. 3 shows the aa alignment of PANEC-1 and PANEC-2 with other humanchemokines of the C-C family. Alignments shown were produced using themultisequence alignment program of DNASTAR software (DNASTAR Inc,Madison Wis.) (Majority=SEQ ID NO:5; MIP 1α=SEQ ID NO:6; MIP 1β=SEQ IDNO:7; RANTES=SEQ ID NO:8; MCP-1=SEQ ID NO:9; MCP-2 =SEQ ID NO:10;MCP-3=SEQ ID NO:11).

FIG. 4 displays an analysis of PANEC-1 hydrophobicity based on thepredicted amino acid sequence and composition.

FIG. 5 displays an analysis of PANEC-2 hydrophobicity based on thepredicted amino acid sequence and composition.

FIG. 6 shows a relatedness tree of human C-C chemokines. Thephylogenetic tree was generated by phylogenetic tree program of DNASTARsoftware using the Clustal method with the PAM250 residue weight table.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, “pancreas expressed chemokines” or PANECs, refer topolypeptides, naturally occurring PANECs or active fragments thereof,which are encoded by mRNAs transcribed from the cDNAs of Seq ID No:1 andSeq ID No:3.

“Active” refers to those forms of PANEC which retain the biologic and/orimmunologic activities of any naturally occurring PANEC.

“Naturally occurring PANEC” refers to PANECs produced by human cellsthat have not been genetically engineered and specifically contemplatesvarious PANECs arising from post-translational modifications of thepolypeptide including but not limited to acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.

“Derivative” refers to polypeptides derived from naturally occurringPANECs by chemical modifications such as ubiquitination, labeling (e.g.,with radionuclides, various enzymes, etc.), pegylation (derivatizationwith polyethylene glycol) or by insertion or substitution by chemicalsynthesis of amino acids such as ornithine, which do not normally occurin human proteins.

“Recombinant variant” refers to any polypeptide differing from naturallyoccurring PANECs by amino acid insertions, deletions, and substitutions,created using recombinant DNA techniques. Guidance in determining whichamino acid residues may be replaced, added or deleted without abolishingactivities of interest, such as cell adhesion and chemotaxis, may befound by comparing the sequence of the particular PANEC with that ofhomologous cytokines and minimizing the number of amino acid sequencechanges made in regions of high homology.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine, i.e., conservative amino acid replacements. “Insertions” or“deletions” are typically in the range of about 1 to 5 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in a PANECmolecule using recombinant DNA techniques and assaying the resultingrecombinant variants for activity.

Where desired, a “signal or leader sequence” can direct the polypeptidethrough the membrane of a cell. Such a sequence may be naturally presenton the polypeptides of the present invention or provided fromheterologous protein sources by recombinant DNA techniques.

A polypeptide “fragment,” “portion,” or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, often at least about 7amino acids, typically at least about 9 to 13 amino acids, and, invarious embodiments, at least about 17 or more amino acids. To beactive, any PANEC polypeptide must have sufficient length to displaybiologic and/or immunologic activity.

An “oligonucleotide” or polynucleotide “fragment”, “portion,” or“segment” is a stretch of nucleotide residues which is long enough touse in polymerase chain reaction (PCR) or various hybridizationprocedures to amplify or simply reveal related parts of mRNA or DNAmolecules.

The present invention includes purified PANEC-1 and PANEC-2 polypeptidesfrom natural or recombinant sources, and cells transformed withrecombinant nucleic acid molecules encoding PANEC-1 and PANEC-2. Variousmethods for the isolation of the PANEC-1 and PANEC-2 polypeptides may beaccomplished by procedures well known in the art. For example, suchpolypeptides may be purified by immunoaffinity chromatography byemploying the antibodies provided by the present invention. Variousother methods of protein purification well known in the art includethose described in Deutscher M (1990) Methods in Enzymology, Vol 182,Academic Press, San Diego; and Scopes R (1982) Protein Purification:Principles and Practice. Springer-Verlag, NYC, both incorporated hereinby reference.

“Recombinant” may also refer to a polynucleotide which encodes PANEC-1or PANEC-2 and is prepared using recombinant DNA techniques. The DNAswhich encode PANEC-1 and PANEC-2 may also include allelic or recombinantvariants and mutants thereof.

“Oligonucleotides” or “nucleic acid probes” are prepared based on thecDNA sequences which encode PANEC-1 and PANEC-2 provided by the presentinvention. Oligonucleotides comprise portions of the DNA sequence havingat least about 15 nucleotides, usually at least about 20 nucleotides.Nucleic acid probes comprise portions of the sequence having fewernucleotides than about 6 kb, usually fewer than about 1 kb. Afterappropriate testing to eliminate false positives, these probes may beused to determine whether mRNAs encoding PANEC-1 and PANEC-2 are presentin a cell or tissue or to isolate similar nucleic acid sequences fromchromosomal DNA as described by Walsh P. S. et al (1992 PCR Methods Appl1:241-250).

Probes may be derived from naturally occurring or recombinant single- ordouble-stranded nucleic acids or be chemically synthesized. They may belabeled by nick translation, Klenow fill-in reaction, PCR or othermethods well known in the art. Probes of the present invention, theirpreparation and/or labeling are elaborated in Sambrook J et al (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York; or Ausubel F. M. et al (1989) Current Protocols in MolecularBiology, John Wiley & Sons, New York City, both incorporated herein byreference.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsmay also be introduced to modify the properties of the polypeptide, tochange ligand-binding affinities, interchain affinities, or polypeptidedegradation or turnover rate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides nucleotide sequences uniquely identifyingnovel chemokines of the C-C family, PANEC-1 and PANEC-2, which arehighly expressed in the pancreas. Because PANEC-1 and PANEC-2 arespecifically expressed in pancreas, the nucleic acids (panec1 andpanec-2), polypeptides (PANEC-1 and PANEC-2) and antibodies to PANEC-1and PANEC-2 are useful in diagnostic assays based on chemokineproduction in cases of inflammation or disease affecting the pancreas.Excessive expression of either PANEC-1 or PANEC-2 can lead to activationof monocytes, macrophages, basophils, eosinophils, T lymphocytes and/orother cells which respond to the chemokines by producing abundantproteases and other molecules which can lead to tissue damage ordestruction. Therefore, a diagnostic test for excess expression ofPANECs can accelerate diagnosis and proper treatment of an abnormalcondition caused by viral or bacterial infections; mechanical injuryassociated with trauma; hereditary diseases affecting pancreatitis;biliary disease; infiltrative diseases such as leukemias and lymphomas;or other physiologic and pathologic problems which affect the functionof the organ.

The nucleotide sequences encoding PANEC-1 and PANEC-2 (or theircomplement) have numerous applications in techniques known to thoseskilled in the art of molecular biology. These techniques include use ashybridization probes, use as oligomers for PCR, use for chromosome andgene mapping, use in the recombinant production of PANEC-1 and PANEC-2,and use in generation of anti-sense DNA or RNA, their chemical analogsand the like. Uses of nucleotides encoding PANEC-1 and PANEC-2 disclosedherein are exemplary of known techniques and are not intended to limittheir use in any technique known to a person of ordinary skill in theart. Furthermore, the nucleotide sequences disclosed herein may be usedin molecular biology techniques that have not yet been developed,provided the new techniques rely on properties of nucleotide sequencesthat are currently known, e.g., the triplet genetic code, specific basepair interactions, etc.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of PANEC-encodingnucleotide sequences, some bearing minimal homology to the nucleotidesequence of any known and naturally occurring gene may be produced. Theinvention has specifically contemplated each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring PANECs, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode PANEC-1 and PANEC-2 and/ortheir variants are preferably capable of hybridizing to the nucleotidesequence of the naturally occurring PANEC genes under stringentconditions, it may be advantageous to produce nucleotide sequencesencoding PANEC-1 and PANEC-2 or their derivatives possessing asubstantially different codon usage. Codons can be selected to increasethe rate at which expression of the peptide occurs in a particularprokaryotic or eukaryotic expression host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encodingPANEC-1 and PANEC-2 and/or their derivatives without altering theencoded amino acid sequence include the production of RNA transcriptshaving more desirable properties, such as a greater half-life, thantranscripts produced from the naturally occurring sequence.

Nucleotide sequences encoding PANEC-1 or PANEC-2 may be joined to avariety of other nucleotide sequences by means of well establishedrecombinant DNA techniques (cf Sambrook J et al. (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).Useful nucleotide sequences for joining to panec include an assortmentof cloning vectors, e.g., plasmids, cosmids, lambda phage derivatives,phagemids, and the like, that are well known in the art. Vectors ofinterest include expression vectors, replication vectors, probegeneration vectors, sequencing vectors, and the like. In general,vectors of interest may contain an origin of replication functional inat least one organism, convenient restriction endonuclease sensitivesites, and selectable markers for the host cell.

Another aspect of the subject invention is to provide for panec-1- orpanec-2-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences encodingPANEC-1 or PANEC-2. Such probes may also be used for the detection ofsimilar chemokine encoding sequences and should preferably contain atleast 50% of the nucleotides from a C-C encoding sequence. Thehybridization probes of the subject invention may be derived from thenucleotide sequences of the SEQ ID NO:1 or SEQ ID NO:3 from genomicsequences including promoters, enhancer elements and introns of therespective naturally occurring panecs. Hybridization probes may belabeled by a variety of reporter groups, including radionuclides such as³²P or ³⁵S, or enzymatic labels such as alkaline phosphatase coupled tothe probe via avidin/biotin coupling systems, and the like.

PCR as described U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188provides additional uses for oligonucleotides based upon the nucleotidesequences which encode either PANEC-1 or PANEC-2. Such probes used inPCR may be of recombinant origin, may be chemically synthesized, or amixture of both and comprise a discrete nucleotide sequence fordiagnostic use or a degenerate pool of possible sequences foridentification of closely related genomic sequences.

Other means of producing specific hybridization probes for panec DNAsinclude the cloning of nucleic acid sequences encoding PANEC-1 andPANEC-2 or PANEC-1 and PANEC-2 derivatives into vectors for theproduction of mRNA probes. Such vectors are known in the art and arecommercially available and may be used to synthesize RNA probes in vitroby means of the addition of the appropriate RNA polymerase as T7 or SP6RNA polymerase and the appropriate radioactively labeled nucleotides.

It is now possible to produce a DNA sequence, or portions thereof,encoding PANEC-1 and PANEC-2 and their derivatives entirely by syntheticchemistry, after which the gene can be inserted into any of the manyavailable DNA vectors using reagents, vectors and cells that are knownin the art at the time of the filing of this application. Moreover,synthetic chemistry may be used to introduce mutations into the panecsequences or any portion thereof.

The nucleotide sequence can be used to construct an assay to detectinflammation or disease associated with abnormal levels of expression ofPANEC-1 or PANEC-2. The nucleotide sequence can be labeled by methodsknown in the art and added to a fluid or tissue sample from a patientunder hybridizing conditions. After an incubation period, the sample iswashed with a compatible fluid which optionally contains a dye (or otherlabel requiring a developer) if the nucleotide has been labeled with anenzyme. After the compatible fluid is rinsed off, the dye is quantitatedand compared with a standard. If the amount of dye is significantlyelevated, the nucleotide sequence has hybridized with the sample, andthe assay indicates the presence of inflammation and/or disease.

The nucleotide sequence for panec-1 or panec-2 can be used to constructhybridization probes for mapping that gene. The nucleotide sequenceprovided herein may be mapped to a chromosome and specific regions of achromosome using well known genetic and/or chromosomal mappingtechniques. These techniques include in situ hybridization, linkageanalysis against known chromosomal markers, hybridization screening withlibraries or flow-sorted chromosomal preparations specific to knownchromosomes, and the like. The technique of fluorescent in situhybridization of chromosome spreads has been described, among otherplaces, in Verma et al (1988) Human Chromosomes: A Manual of BasicTechniques, Pergamon Press, New York City.

Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofpanec on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) can help delimit the region of DNAassociated with that genetic disease. The nucleotide sequence of thesubject invention may be used to detect differences in gene sequencebetween normal and carrier or affected individuals.

Nucleotide sequences encoding PANEC-1 and PANEC-2 may be used to producepurified PANEC-1 and PANEC-2 using well known methods of recombinant DNAtechnology. Among the many publications that teach methods for theexpression of genes after they have been isolated is Goeddel (1990) GeneExpression Technology, Methods and Enzymology, Vol 185, Academic Press,SanuDiego. PANEC-1 and PANEC-2 may be expressed in a variety of hostcells, either prokaryotic or eukaryotic. Host cells may be from the samespecies in which panec nucleotide sequences are endogenous or from adifferent species. Advantages of producing PANEC-1 and PANEC-2 byrecombinant DNA technology include obtaining adequate amounts of theprotein for purification and the availability of simplified purificationprocedures.

Cells transformed with DNA encoding PANEC-1 or PANEC-2 may be culturedunder conditions suitable for the expression of chemokines and recoveryof the protein from the cell culture. PANEC-1 or PANEC-2 produced by arecombinant cell may be secreted or may be contained intracellularly,depending on the particular genetic construction used. In general, it ismore convenient to prepare recombinant proteins in secreted form.Purification steps vary with the production process and the particularprotein produced.

In addition to recombinant production, fragments of PANEC-1 or PANEC-2may be produced by direct peptide synthesis using solid-phase techniques(cf Stewart et al (1969) Solid-Phase Peptide Synthesis, W. H. FreemanCo, San Francisco; Merrifield J (1963) J Am Chem Soc 85:2149-2154. Invitro protein synthesis may be performed using manual techniques or byautomation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Foster City, Calif.) inaccordance with the instructions provided by the manufacturer. Variousfragments of PANEC-1 and PANEC-2 may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

PANEC-1 or PANEC-2 for antibody induction does not require biologicalactivity; however, the protein must be immunogenic. Peptides used toinduce specific antibodies may have an amino acid sequence consisting ofat least five amino acids, preferably at least 10 amino acids. Theyshould mimic a portion of the amino acid sequence of the protein and maycontain the entire amino acid sequence of a small naturally occurringmolecules like PANEC-1 and PANEC-2. Short stretches of PANEC-1 orPANEC-2 amino acids may be fused with those of another protein such askeyhole limpet hemocyanin and the chimeric molecule used for antibodyproduction.

Antibodies specific for PANEC-1 or PANEC-2 may be produced byinoculation of an appropriate animal with the polypeptide or anantigenic fragment. An antibody is specific for PANEC-1 or PANEC-2 if itis produced against an epitope of the polypeptide and binds to at leastpart of the natural or recombinant protein. Antibody production includesnot only the stimulation of an immune response by injection intoanimals, but also analogous steps in the production of syntheticantibodies or other specific-binding molecules such as the screening ofrecombinant immunoglobulin libraries (cf Orlandi R et al (1989) PNAS86:3833-3837, or Huse W. D. et al (1989) Science 256:1275-1281) or thein vitro stimulation of lymphocyte populations. Current technology(Winter G and Milstein C (1991) Nature 349:293-299) provides for anumber of highly specific binding reagents based on the principles ofantibody formation. These techniques may be adapted to produce moleculesspecifically binding PANECs.

An additional embodiment of the subject invention is the use of PANEC-1or PANEC-2 specific antibodies, inhibitors, receptors or their analogsas bioactive agents to treat inflammation or disease of the pancreasincluding, but not limited to viral or bacterial infections; mechanicalinjury associated with trauma; hereditary diseases affectingpancreatitis; biliary disease; infiltrative diseases such as leukemiasand lymphomas; or other physiologic and pathologic problems which affectthe function of the organ.

Bioactive compositions comprising agonists, antagonists, receptors orinhibitors of PANEC-1 or PANEC-2 may be administered in a suitabletherapeutic dose determined by any of several methodologies includingclinical studies on mammalian species to determine maximal tolerabledose and on normal human subjects to determine safe dose. Additionally,the bioactive agent may be complexed with a variety of well establishedcompounds or compositions which enhance stability or pharmacologicalproperties such as half-life. It is contemplated that the therapeutic,bioactive composition may be delivered by intravenous infusion into thebloodstream or any other effective means which could be used fortreating problems of the pancreas.

The examples below are provided to illustrate the subject invention.These examples are provided by way of illustration and are not includedfor the purpose of limiting the invention.

EXAMPLES

I Isolation of mRNA and Construction of cDNA Libraries

The panec-1 and panec-2 cDNA sequences were identified among thesequences comprising the human pancreas library. The normal pancreasused for this library was obtained from the Keystone Skin Bank,International Institute for the Advancement of Medicine (Exton, Pa.).Normal pancreas tissue from a 56 year old Caucasian male (Lot HDS330)wasflash frozen, ground in a mortar and pestle, and lyzed immediately inbuffer containing guanidinium isothiocyanate. Lysis was followed byseveral phenol chloroform extractions and ethanol precipitation. Poly A⁺RNA was isolated using biotinylated oligo d(T) primer and streptavidincoupled to a paramagnetic particle (Promega Corp, Madison Wis.) and sentto Stratagene (11011 North Torrey Pines Road, La Jolla, Calif. 92037).

An alternate method of purifying phagemid has recently become available.It utilizes the MINIPREP kit (Catalog No. 77468, available from AdvancedGenetic Technologies Corp., 19212 Orbit Drive, Gaithersburg, Md.). Thiskit is in the 96-well format and provides enough reagents for 960purifications. Each kit is provided with a recommended protocol, whichhas been employed except for the following changes. First, the 96 wellsare each filled with only 1 ml of sterile terrific broth withcarbenicillin at 25 mg/L and glycerol at 0.4%. After the wells areinoculated, the bacteria are cultured for 24 hours and lysed with 60 μlof lysis buffer. A centrifugation step (2900 rpm for 5 minutes) isperformed before the contents of the block are added to the primaryfilter plate. The optional step of adding isopropanol to TRIS buffer isnot routinely performed. After the last step in the protocol, samplesare transferred to a Beckman 96-well block for storage.

Stratagene prepared the cDNA library using oligo d(T) priming. Syntheticadapter oligonucleotides were ligated onto the cDNA molecules enablingthem to be inserted into the UNI-ZAP vector system (Stratagene). Thisallowed high efficiency unidirectional (sense orientation) lambdalibrary construction and the convenience of a plasmid system withblue/white color selection to detect clones with cDNA insertions.

The quality of the cDNA library was screened using DNA probes, and then,the PBLUESCRIPT phagemid (Stratagene) was excised. This phagemid allowsthe use of a plasmid system for easy insert characterization,sequencing, site-directed mutagenesis, the creation of unidirectionaldeletions and expression of fusion polypeptides. Subsequently, thecustom-constructed library phage particles were infected into E. colihost strain XL1-BLUE (Stratagene). The high transformation efficiency ofthis bacterial strain increases the probability that the cDNA librarywill contain rare, under-represented clones. Alternative unidirectionalvectors might include, but are not limited to, pcDNAI (Invitrogen) andpSH1ox-1 (Novagen).

II Isolation of cDNA Clones

The phagemid forms of individual cDNA clones were obtained by the invivo excision process, in which XL1-BLUE was coinfected with an f1helper phage. Proteins derived from both lambda phage and f1helper phageinitiated new DNA synthesis from defined sequences on the lambda targetDNA and create a smaller, single-stranded circular phagernid DNAmolecule that includes all DNA sequences of the pBluescript plasmid andthe cDNA insert. The phagemid DNA was released from the cells andpurified, then used to re-infect fresh bacterial host cells (SOLR,Stratagene Inc), where the double-stranded phagemid DNA was produced.Because the phagemid carries the gene for β-lactamase, the newlytransformed bacteria were selected on medium containing ampicillin.

Phagemid DNA was purified using the QIAWELL-8 plasmid purificationsystem from QIAGEN DNA purification system (QIAGEN Inc, 9259 Eton Ave,Chatsworth, Calif. 91311). This technique provides a rapid and reliablehigh-throughput method for lysing the bacterial cells and isolatinghighly purified phagemid DNA. The DNA eluted from the purification resinwas suitable for DNA sequencing and other analytical manipulations.

An alternate method of purifying phagemid has recently become available.It utilizes the MINIPREP kit (Catalog No. 77468, available from AdvancedGenetic Technologies Corp., 19212 Orbit Drive, Gaithersburg, Md.). Thiskit is in the 96-well format and provides enough reagents for 960purifications. Each kit is provided with a recommended protocol, whichhas been employed except for the following changes. First, the 96 wellsare each filled with only 1 ml of sterile terrific broth withcarbenicillin at 25 mg/L and glycerol at 0.4%. After the wells areinoculated, the bacteria are cultured for 24 hours and lysed with 60 μlof lysis buffer. A centrifugation step (2900 rpm for 5 minutes) isperformed before the contents of the block are added to the primaryfilter plate. The optional step of adding isopropanol to TRIS buffer isnot routinely performed. After the last step in the protocol, samplesare transferred to a Beckman 96-well block for storage.

III Sequencing of cDNA Clones

The cDNA inserts from random isolates of the human pancreas library weresequenced in part. Methods for DNA sequencing are well known in the art.Conventional enzymatic methods employed DNA polymerase Klenow fragment,SEQUENASE (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest. Methods have been developed for the use of bothsingle- and double-stranded templates. The chain termination reactionproducts were electrophoresed on urea-acrylamide gels and detectedeither by autoradiography (for radionuclide-labeled precursors) or byfluorescence (for fluorescent-labeled precursors). Recent improvementsin mechanized reaction preparation, sequencing and analysis using thefluorescent detection method have permitted expansion in the number ofsequences that can be determined per day (using machines such as theCatalyst 800 and the Applied Biosystems 373 DNA sequencer).

IV Homology Searching of cDNA Clones and Deduced Proteins

Each sequence so obtained was compared to sequences in GenBank using asearch algorithm developed by Applied Biosystems Inc. and incorporatedinto the INHERIT 670 Sequence Analysis System. In this algorithm,Pattern Specification Language (developed by TRW Inc.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT 670 sequence analysis system in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

The nucleotide and amino acid sequences for the entire coding region ofthe the pancreas expressed chemokines, PANEC-1 and PANEC-2, claimed inthis invention are shown in FIG. 1.

V Identification and Full Length Sequencing of the Genes

From all of the randomly picked and sequenced clones of the humanpancreas library, the panec sequences were homologous to but clearlydifferent from one another and from any known C-C chemokine molecule.The complete nucleotide sequences for panec-1 and panec-2 weretranslated, and the in-frame translations, as identified, are shown inFIGS. 1 and 2, respectively. When all three possible predictedtranslations of the sequence were searched against protein databasessuch as SwissProt and PIR, no exact matches were found to the possibletranslations of panec-1 or panec-2. FIG. 3 shows the comparison ofPANEC-1 and PANEC-2 amino acid sequences with other B chemokinemolecules. The substantial regions of homology among these moleculeswhich includes the definitive C-C motif are shaded. Hydrophobicity plotsfor PANEC-1 and PANEC-2 are shown as FIGS. 4 and 5, respectively. Thephylogenetic analysis (FIG. 6) shows how closely panec-1 and panec-2 arerelated to one another and to other well characterized human C-Cchemokines. The most related of these molecules cluster together at theright hand side of the figure.

VI Antisense Analysis

Knowledge of the correct, complete cDNA sequences of novel expressedchemokine genes will enable their use in antisense technology in theinvestigation of gene function. Either oligonucleotides, genomic or cDNAfragments comprising the antisense strand of panec-1 or panec-2 can beused either in vitro or in vivo to inhibit expression of the specificprotein. Such technology is now well known in the art, and probes can bedesigned at various locations along the nucleotide sequences. Bytreatment of cells or whole test animals with such antisense sequences,the gene of interest can be effectively turned off. Frequently, thefunction of the gene can be ascertained by observing behavior at thecellular, tissue or organismal level (e.g. lethality, loss ofdifferentiated function, changes in morphology, etc.).

In addition to using sequences constructed to interrupt transcription ofthe open reading frame, modifications of gene expression can be obtainedby designing antisense sequences to intron regions, promoter/enhancerelements, or even to trans-acting regulatory genes. Similarly,inhibition can be achieved using Hogeboom base-pairing methodology, alsoknown as “triple helix” base pairing.

VII Expression of PANEC-1 and PANEC-2

Expression of panec-1 and panec-2 may be accomplished by subcloning thecDNAs into appropriate expression vectors and transfecting the vectorsinto an appropriate expression hosts. In this particular case, thecloning vector previously used for the generation of the tissue libraryalso provide for direct expression of the included panec-1 and panec-2sequences in E. coli. Upstream of the cloning site, this vector containsa promoter for β-galactosidase, followed by sequence containing theamino-terminal Met and the subsequent 7 residues of β-galactosidase.Immediately following these eight residues is an engineeredbacteriophage promoter useful for artificial priming and transcriptionand a number of unique restriction sites, including Eco RI, for cloning.

Induction of the isolated, transfected bacterial strain with IPTG usingstandard methods will produce a fusion protein corresponding to thefirst seven residues of β-galactosidase, about 15 residues of “linker”,and the peptide encoded within the cDNA. Since cDNA clone inserts aregenerated by an essentially random process, there is one chance in threethat the included cDNA will lie in the correct frame for propertranslation. If the cDNA is not in the proper reading frame, it can beobtained by deletion or insertion of the appropriate number of bases bywell known methods including in vitro mutagenesis, digestion withexonuclease III or mung bean nuclease, or oligonucleotide linkerinclusion.

Panec-1 or panec-2 cDNA can be shuttled into other vectors known to beuseful for expression of protein in specific hosts. Oligonucleotideamplimers containing cloning sites as well as a segment of DNAsufficient to hybridize to stretches at both ends of the target cDNA (25bases) can be synthesized chemically by standard methods. These primerscan then used to amplify the desired gene segments by PCR. The resultingnew gene segments can be digested with appropriate restriction enzymesunder standard conditions and isolated by gel electrophoresis.Alternately, similar gene segments can be produced by digestion of thecDNA with appropriate restriction enzymes and filling in the missinggene segments with chemically synthesized oligonucleotides. Segments ofthe coding sequence from more than one gene can be ligated together andcloned in appropriate vectors to optimize expression of recombinantsequence.

Suitable expression hosts for such chimeric molecules include but arenot limited to mammalian cells such as Chinese Hamster Ovary (CHO) andhuman 293 cells, insect cells such as Sf9 cells, yeast cells such asSaccharomvces cerevisiae, and bacteria such as E. coli. For each ofthese cell systems, a useful expression vector may also include anorigin of replication to allow propagation in bacteria and a selectablemarker such as the β-lactamase antibiotic resistance gene to allowselection in bacteria. In addition, the vectors may include a secondselectable marker such as the neomycin phosphotransferase gene to allowselection in transfected eukaryotic host cells. Vectors for use ineukaryotic expression hosts may require RNA processing elements such as3′ polyadenylation sequences if such are not part of the cDNA ofinterest.

Additionally, the vector may contain promoters or enhancers whichincrease gene expression. Such promoters are host specific and includeMMTV, SV40, or metallothionine promoters for CHO cells; trp, lac, tac orT7 promoters for bacterial hosts, or alpha factor, alcohol oxidase orPGH promoters for yeast. Transcription enhancers, such as the roussarcoma virus (RSV) enhancer, may be used in mammalian host cells. Oncehomogeneous cultures of recombinant cells are obtained through standardculture methods, large quantities of recombinantly produced PANEC-1 andPANEC-2 can be recovered from the conditioned medium and analyzed usingchromatographic methods known in the art.

VIII Isolation of Recombinant PANEC-1 and PANEC-2

PANEC may be expressed as a chimeric protein with one or more additionalpolypeptide domains added to facilitate protein purification. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp., SeattleWash.). The inclusion of a cleavable linker sequence such as Factor XAor enterokinase(Invitrogen, San Diego Calif.) between the purificationdomain and the panec sequence may be useful to facilitate expression ofPANEC.

IX Production of PANEC-1 and PANEC-2 Specific Antibodies

Two approaches are utilized to raise antibodies to PANEC-1 and PANEC-2,and each approach is useful for generating either polyclonal ormonoclonal antibodies. In one approach, denatured protein from thereverse phase HPLC separation is obtained in quantities up to 75 mg.This denatured protein can be used to immunize mice or rabbits usingstandard protocols; about 100 micrograms are adequate for immunizationof a mouse, while up to 1 mg might be used to immunize a rabbil. Foridentifying mouse hybridomas, the denatured protein can beradioiodinated and used to screen potential murine B-cell hybridomas forthose which produce antibody. This procedure requires only smallquantities of protein, such that 20 mg would be sufficient for labelingand screening of several thousand clones.

In the second approach, the amino acid sequence of PANEC-1 or PANEC-2,as deduced from translation of the cDNA, is analyzed to determineregions of high immunogenicity. Oligopeptides comprising appropriatehydrophilic regions, as shown in FIGS. 4 and 5, are synthesized and usedin suitable immunization protocols to raise antibodies. Analysis toselect appropriate epitopes is described by Ausubel F. M. et al (1989,Current Protocols in Molecular Biology, John Wiley & Sons, New YorkCity). The optimal amino acid sequences for immunization are usually atthe C-terminus, the N-terminus and those intervening, hydrophilicregions of the polypeptide which are likely to be exposed to theexternal environment when the protein is in its natural conformation.

Typically, selected peptides, about 15 residues in length, aresynthesized using an Applied Biosystems peptide synthesizer Model 431Ausing fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH,Sigma) by reaction with M -maleimidobenzoyl-N- hydroxysuccinimide ester(MBS; cf. Ausubel FM et al, supra). If necessary, a cysteine may beintroduced at the N-terminus of the peptide to permit coupling to KLH.Rabbits are immunized with the peptide-KLH complex in complete Freund'sadjuvant. The resulting antisera are tested for antipeptide activity bybinding the peptide to plastic, blocking with 1% BSA, reacting withantisera, washing and reacting with labeled (radioactive orfluorescent), affinity purified, specific goat anti-rabbit IgG.

Hybridomas may also be prepared and screened using standard techniques.Hybridomas of interest are detected by screening with labeled PANEC-1 orPANEC-2 to identify those fusions producing the monoclonal antibody withthe desired specificity. In a typical protocol, wells of plates (FAST;Becton-Dickinson, Palo Alto, Calif.) are coated with affinity purified,specific rabbit-anti-mouse (or suitable anti-species Ig) antibodies at10 mg/ml. The coated wells are blocked with 1% BSA, washed and exposedto supernatants from hybridomas. After incubation the wells are exposedto labeled PANEC-1 or PANEC-2 at 1 mg/ml. Clones producing antibodieswill bind a quantity of labeled PANEC-1 or PANEC-2 which is detectableabove background. Such clones are expanded and subjected to 2 cycles ofcloning at limiting dilution (1 cell/3 wells). Cloned hybridomas areinjected into pristame mice to produce ascites, and monoclonal antibodyis purified from mouse ascitic fluid by affinity chromatography onProtein A. Monoclonal antibodies with affinities of at least 10⁸M⁻¹,preferably 10⁹ to 10¹⁰ or stronger, will typically be made by standardprocedures as described in Harlow and Lane (1988) Antibodies: ALaboratory Manual. Cold Spring Harbor Laboratory New York; and in Goding(1986) Monoclonal Antibodies: Principles and Practice, Academic Press,New York City, both incorporated herein by reference.

X Diagnostic Test Using PANEC-1 and PANEC-2 Specific Antibodies

Particular PANEC-1 or PANEC-2 antibodies are useful for the diagnosis ofprepathologic conditions, and chronic or acute diseases which arecharacterized by differences in the amount or distribution of PANEC-1 orPANEC-2, respectively. To date, PANEC-1 and PANEC-2 has only been foundin the human pancreas library and is thus specific for abnormalities orpathologies which affect the pancreas.

Diagnostic tests for PANEC include methods utilizing the antibody and alabel to detect PANEC in human body fluids, tissues or extracts of suchtissues. The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a substance which provides for a detectable signal.A wide variety of labels and conjugation techniques are known and havebeen reported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent agents, chemiluminescent agents, magneticparticles and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also, recombinant immunoglobulins may beproduced as shown in U.S. Pat. No. 4,816,567, incorporated herein byreference.

A variety of protocols for measuring soluble or membrane-bound PANEC-1or PANEC-2, using either polyclonal or monoclonal antibodies specificfor the respective protein are known in the art. Examples includeenzyme-linked immunosorbent assay (ELISA), radioimnmunoassay (RIA) andfluorescent activated cell sorting (FACS). A two-site monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on PANEC-1 or PANEC-2 is preferred, but acompetitive binding assay may be employed. These assays are described,among other places, in Maddox, D. E. et al (1983, J Exp Med 158:1211).

XI Purification of Native PANEC-1 and PANEC-2 Using Specific Antibodies

Native or recombinant PANEC-1 or PANEC-2 can be purified byimmunoaffinity chromatography using antibodies specific for eitherPANEC-1 or PANEC-2, respectively. In general, an immunoaffinity columnis constructed by covalently coupling the anti- PANEC-1 or PANEC-2antibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such immunoaffinity columns were utilized in the purification of PANEC-1and PANEC-2 by preparing a fraction from cells containing PANEC-1 orPANEC-2 in a soluble form. This preparation was derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PANEC-1 or PANEC-2containing a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PANEC-1 or PANEC-2 -containing preparation was passed over theimmunoaffinity column, and the column was washed under conditions thatallow the preferential absorbance of chemokines (eg, high ionic strengthbuffers in the presence of detergent). Then, the column was eluted underconditions that disrupt antibody/chemokine binding (e.g., a buffer of pH2-3 or a high concentration of a chaotrope such as urea or thiocyanateion), and PANEC-1 or PANEC-2 was collected.

XII PANEC-1 and PANEC-2 Induced Chemotaxis or Cell Activation

The chemotactic activities of PANEC-1 and PANEC-2 were measured in48-well microchemotaxis chambers (Falk W. R. et al (1980) J ImmunolMethods 33:239). In each well, two compartments are separated by afilter that allows the passage of cells in response to a chemicalgradient. Cell culture medium such as RPMI 1640 (Sigma, St. Louis Mo.)containing the expressed chemokine is placed on one side of a filter,usually polycarbonate, and cells suspended in the same media are placedon the opposite side of the filter. Sufficient incubation time isallowed for the cells to traverse the filter in response to theconcentration gradient across the filter. Filters are recovered fromeach well, and cells adhering to the side of the filter facing thechemokine are typed and quantified.

The specificity of the chemoattraction is determined by performing thechemotaxis assay on specific populations of cells. First, blood cellsobtained from venipuncture are fractionated by density gradientcentrifugation and the chemotactic activity of PANEC-1 or PANEC-2 istested on enriched populations of neutrophils, peripheral bloodmononuclear cells, monocytes and lymphocytes. Optionally, such enrichedcell populations are further fractionated using CD8+ and CD4+ specificantibodies for negative selection of CD4+ and CD8+ enriched T-cellpopulations, respectively.

Another assay elucidates the chemotactic effect of PANEC-1 or PANEC-2 onactivated T-cells. There, unfractionated T-cells or fractionated T-cellsubsets are cultured for 6 to 8 hours in tissue culture vessels coatedwith CD-3 antibody. After this CD-3 activation, the chemotactic activityof PANEC-1 or PANEC-2 is tested as described above. Many other methodsfor obtaining enriched cell populations are known in the art.

Some chemokines also produce a non-chemotactic cell activation ofneutrophils and monocytes. This is tested via standard measures ofneutrophil activation such as actin polymerization, increase inrespiratory burst activity, degranulation of the azurophilic granule andmobilization of Ca⁺⁺ as part of the signal transduction pathway. Theassay for mobilization of Ca⁺⁺ involves preloading neutrophils with afluorescent probe whose emission characteristics have been altered byCa⁺⁺ binding. When the cells are exposed to an activating stimulus, Ca⁺⁺flux is determined by observation of the cells in a fluorometer. Themeasurement of Ca⁺⁺ mobilization has been described in Grynkievicz G etal. (1985) J Biol Chem 260:3440, and McColl S et al. (1993) J Immunol150:4550-4555, incorporated herein by reference.

Degranulation and respiratory burst responses are also measured inmonocytes (Zachariae COC et al. (1990) J Exp Med 171: 2177-82). Furthermeasures of monocyte activation are regulation of adhesion moleculeexpression and cytokine production (Jiang Y et al. (1992) J Immunol148:2423-8). Expression of adhesion molecules also varies withlymphocyte activation (Taub D et al. (1993) Science 260: 355-358).

XIII Drug Screening

This invention is particularly useful for screening compounds by usingPANEC-1 or PANEC-2 polypeptide or binding fragments thereof in any of avariety of drug screening techniques. The chemokine polypeptide orfragment employed in such a test may either be free in solution, affixedto a solid support, borne on a cell surface or located intracellularly.One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant nucleic acidsexpressing the polypeptide or fragment. Drugs are screened against suchtransformed cells in competitive binding assays. Such cells, either inviable or fixed form, can be used for standard binding assays. One maymeasure, for example, the formation of complexes between PANEC-1 orPANEC-2 and the agent being tested. Alternatively, one can examine thediminution in complex formation between PANEC-1 or PANEC-2 and itstarget cell, for example, a monocyte caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect inflammation and disease. Thesemethods comprise contacting such an agent with a PANEC-1 or PANEC-2polypeptide or fragment thereof and assaying (i) for the presence of acomplex between the agent and the PANEC-1 or PANEC-2 polypeptide orfragment, or (ii) for the presence of a complex between the PANEC-1 orPANEC-2 polypeptide or fragment and the cell, by methods well known inthe art. In such competitive binding assays, the chemokine polypeptideor fragment is typically labeled. After suitable incubation, freePANEC-1 or PANEC-2 polypeptide or fragment is separated from thatpresent in bound form, and the amount of free or uncomplexed label is ameasure of the ability of the particular agent to bind to PANEC-1 orPANEC-2 or to interfere with the PANEC-1 or PANEC-2 and agent complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the PANEC-1 or PANEC-2polypeptides and is described in detail in European Patent Application84/03564, published on Sep. 13, 1984, incorporated herein by reference.Briefly stated, large numbers of different small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with PANEC-1 or PANEC-2polypeptide and washed. Bound PANEC-1 or PANEC-2 polypeptide is thendetected by methods well knovn in the art. Purified PANEC-1 or PANEC-2can also be coated directly onto plates for use in the aforementioneddrug screening techniques. In addition, non-neutralizing antibodies canbe used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PANEC-1 orPANEC-2 specifically compete with a test compound for binding tochemokine polypeptides or fragments thereof. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with PANEC-1 or PANEC-2.

XIV. Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact, e.g., agonists, antagonists, or inhibitors. Any ofthese examples can be used to fashion drugs which are more active orstable forms of the polypeptide or which enhance or interfere with thefunction of a polypeptide in vivo (cf Hodgson J (1991) Bio/Technology9:19-21, incorporated herein by reference).

In one approach, the three-dimensional structure of a protein ofinterest, or of a protein-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous chemokine-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton S and Wells J. A. (1992 Biochemistry31:7796- 7801) or which act as inhibitors, agonists, or antagonists ofnative peptides as shown by Athauda S. B. et al (1993 J Biochem113:742-746), incorporated herein by reference.

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amount of polypeptide maybe made available to perform such analytical studies as X-raycrystallography. In addition, knowledge of the PANEC amino acid sequenceprovided herein will provide guidance to those employing computermodeling techniques in place of or in addition to x-ray crystallography.

XV Identification of PANEC-1 and PANEC-2 Receptors

Purified PANEC-1 and PANEC-2 are useful for characterization andpurification of specific cell surface receptors and other bindingmolecules. Cells which respond to PANEC-1 and PANEC-2 by chemotaxis orother specific responses are likely to express a receptor for PANEC-1and PANEC-2, respectively. Radioactive labels may be incorporated intoPANEC-1 and PANEC-2 by various methods known in the art. A preferredembodiment is the labeling of primary amino groups in PANEC-1 andPANEC-2 with ¹²⁵I Bolton-Hunter reagent (Bolton, A. E. and Hunter, W. M.(1973) Biochem J 133: 529), which has been used to label otherchemokines without concomitant loss of biological activity (Hebert C. A.et al (1991) J Biol Chem 266: 18989; McColl S et al (1993) J Immunol150:45504555). Receptor-bearing cells are incubated with the labeledchemokine molecule. The cells are then washed to removed unboundchemokine, and receptor-bound labeled molecule is quantified. The dataobtained using different concentrations of PANEC-1 or PANEC-2 are usedto calculate values for the number and affinity of receptors.

Labeled PANEC-1 or PANEC-2 is also useful as a reagent for purificationof its specific receptor. In one embodiment of affinity purification,the chemokine is covalently coupled to a chromatography column.Receptor-bearing cells are extracted, and the extract is passed over thecolumn. The receptor binds to the column by virtue of its biologicalaffinity for either PANEC-1 or PANEC-2. The receptor is recovered fromthe column and subjected to N-terminal protein sequencing. This aminoacid sequence is then used to design degenerate oligonucleotide probesfor cloning the receptor gene.

In an alternate method, mRNA is obtained from receptor-bearing cells andmade into a cDNA library. The library is transfected into a populationof cells, and those cells expressing the receptor are selected usingfluorescently labeled PANEC-1 or PANEC-2. The PANEC-1 or PANEC-2specific receptor is identified by recovering and sequencing recombinantDNA from highly labeled cells.

In another alternate method, antibodies are raised against the surfaceof receptor- bearing cells, specifically monoclonal antibodies. Themonoclonal antibodies are screened to identify those which inhibit thebinding of labeled PANEC-1 or PANEC-2. These monoclonal antibodies arethen used in affinity purification or expression cloning of thereceptor.

Soluble receptors or other soluble binding molecules are identified in asimilar manner. Labeled PANEC-1 or PANEC-2 is incubated with extracts orother appropriate materials derived from the pancreas. After incubation,PANEC-1 or PANEC-2 complexes (which are larger than the size of purifiedthe purified chemokine molecule) are identified by a sizing techniquesuch as size exclusion chromatography or density gradient centrifugationand are purified by methods known in the art. The soluble receptors orbinding protein(s) are subjected to N-terminal sequencing to obtaininformation sufficient for database identification, if the solubleprotein is known, or for cloning, if the soluble protein is unknown.

XVI Use and Administration of PANEC-1 and PANEC-2

Antibodies, inhibitors, receptors or antagonists of PANEC-1 and PANEC-2(or other treatments for excessive chemokine production, hereinafterabbreviated TEC), can provide different effects when administeredtherapeutically. TECs will be formulated in a nontoxic, inert,pharmaceutically acceptable aqueous carrier medium preferably at a pH ofabout 5 to 8, more preferably 6 to 8, although the pH may vary accordingto the characteristics of the antibody, inhibitor, receptor orantagonist being formulated and the condition to be treated.Characteristics of TEC include solubility of the molecule, half-life andantigenicity/immuno-genicity; these and other characteristics may aid indefining an effective carrier. Native human proteins are preferred asTECs, but organic or synthetic molecules resulting from drug screens maybe equally effective in particular situations.

TECs may be delivered by known routes of administration including butnot limited to topical creams and gels; transmucosal spray and aerosol,transdermal patch and bandage; injectable, intravenous and lavageformulations; and orally administered liquids and pills, particularlyformulated to resist stomach acid and enzymes. The particularformulation, exact dosage, and route of administration will bedetermined by the attending physician and will vary according to eachspecific situation.

Such determinations are made by considering multiple variables such asthe condition to be treated, the TEC to be administered, and thepharmacokinetic profile of the particular TEC. Additional factors whichmay be taken into account include disease state (e.g. severity) of thepatient, age, weight, gender, diet, time of administration, drugcombination, reaction sensitivities, and tolerance/response to therapy.Long acting TEC formulations might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular TEC.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.It is anticipated that different formulations will be effective fordifferent TECs and that administration targeting the pancreas maynecessitate delivery in a manner different from that to another organ ortissue.

It is contemplated that conditions or diseases of the pancreas whichactivate monocytes, macrophages, basophils, eosinophils or otherleukocytes may precipitate damage that is treatable with TECs. Theseconditions or diseases may be specifically diagnosed by the testsdiscussed above, and such testing should be performed in suspected casesof viral or bacterial infections; mechanical injury associated withtrauma; hereditary diseases affecting pancreatitis; biliary disease;infiltrative diseases such as leukemias and lymphomas; or otherphysiologic and pathologic problems which affect the function of theorgan.

All publications and patents mentioned in the above specification areherein incorporated by reference. The foregoing written specification isconsidered to be sufficient to enable one skilled in the art to practicethe invention. Indeed, various modifications of the above describedmodes for carrying out the invention which are obvious to those skilledin the field of molecular biology or related fields are intended to bewithin the scope of the following claims.

1. (canceled)
 2. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) SEQ ID NO:2 or SEQID NO:4; and (b) a variant of SEQ ID NO:2 or SEQ ID NO:4 that includesone of: (i) a conservative amino acid substitution in SEQ ID NO:2 or SEQID NO:4; (ii) an insertion of from 1-5 amino acids in SEQ ID NO:2 or SEQID NO:4; and (iii) a deletion of from 1-5 amino acids in SEQ ID NO:2 orSEQ ID NO:4; wherein the variant has chemokine activity.
 3. The isolatedpolypeptide of claim 2, comprising an amino acid sequence of SEQ ID NO:2or SEQ ID NO:4.
 4. The isolated polypeptide of claim 2, furthercomprising a signal or leader sequence.
 5. A composition comprising thepolypeptide of claim 2 and a pharmaceutically acceptable excipient.
 6. Amethod of screening a compound for effectiveness as an agonist of thepolypeptide of claim 2, the method comprising: a) exposing a samplecomprising the polypeptide of claim 2 to the compound, and b) detectingagonist activity in the sample.
 7. A method of screening a compound foreffectiveness as an antagonist of the polypeptide of claim 2, the methodcomprising: a) exposing a sample comprising the polypeptide of claim 2to the compound, and b) detecting antagonist activity in the sample. 8.A method of screening for a compound that specifically binds to thepolypeptide of claim 2, the method comprising: a) combining thepolypeptide of claim 2 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 2 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim 2.